1. Field
The invention is in the field of staining cytology and histology specimens.
2. State of the Art
The field of pathology involves the microscopic examination of fixed cytology specimens (individual cells in a smear or cell block) and histology specimens (cell aggregates that form a structure with a specific function). These specimens are examined to determine if tissue is normal or diseased. A specimen is processed and applied to a microscope slide and then stained to make the normally transparent cells brilliantly colored for easier observation and to distinguish the various cellular elements which have differing affinities for the various stains.
Cytology specimens are prepared by smearing a sample onto a microscope slide and wet fixing with an alcohol solution which usually contains polyethylene glycol [PEG]. Cytocentrifugation may also be used to deposit cells onto slides from dilute cell suspensions, e.g., body fluids. The PEG is removed prior to staining by exposing the specimen to a washing-dehydrating reagent. The washing-dehydrating reagents employed in staining may include alcohols of one to three carbon atoms. Typically ethanol is employed. However, it is possible to use compounds such as glycols, ketones, esters and ethers of from two to about five carbon atoms, or mixtures thereof.
Specimen preparation for histology and cytology samples require different procedures. For histology specimens, the tissue pieces are fixed in a suitable fixative, typically formalin, and embedded in melted paraffin wax. The wax block is then cut on a microtome to yield a thin slice of paraffin containing the tissue. The specimen slice is then applied to a microscope slide, air dried, and heated to cause the specimen to adhere to the glass slide. Residual paraffin is then dissolved with a suitable solvent, typically xylene, toluene, or others. These so-called deparaffinizing solvents are then removed with a washing-dehydrating type reagent prior to staining. Alternatively, slices may be prepared from frozen specimens, fixed briefly in 10% formalin, then infused with dehydrating reagent. Consequently, a common step for both cytology and histology specimens is the removal of the dehydrating reagent prior to staining with an aqueous stain.
The usual method for staining cytology samples is the Papanicolaou staining technique, usually a progressive stain, while the most common method for histologic staining is the hematoxylineosin [H&E] staining technique which is typically a regressive stain. After the initial preparation of the specimen, these methods share common steps and are quite similar in their characteristics.
The typical technique for performing Papanicolaou and H&E staining is the immersion (dip) technique of staining either manually or with the aid of automated equipment. Immersion Papanicolaou staining consists of the following steps with the steps that are omitted from the H&E method indicated:
(a) Removing dehydrating reagent from a specimen affixed to a microscope slide and hydrating the specimen by soaking in water; PA0 (b) Applying hematoxylin for staining the cell nuclei in the specimen; PA0 (c) Removing excess hematoxylin by rinsing with water [For a regressive hematoxylin stain, the water rinse is usually followed by rinsing with an acid-alcohol followed by rinsing with water to remove the acid-alcohol]; PA0 (d) Contacting the slide with a concentrated solution having a pH above 5.0 to turn the hematoxylin blue [bluing solution]; PA0 (e) Removing the bluing solution by rinsing with water; PA0 (f) [Omitted from H&E method] Staining cytoplasmic elements in the specimen with an alcoholic solution of the dye, orange G, that stains keratinized tissue yellow to orange; PA0 (g) [Omitted from H&E method] Washing away excess orange G by rinsing with a dehydrating reagent; PA0 (h) Staining other cytoplasmic elements with an alcoholic solution of eosin Y, a red stain, and light green or fast green, [Together, this stain combination is the Papanicolaou EA stain. The Papanicolaou EA stain differentiates between superficial cells which stain red and intermediate cells which stain blue to green. The green stain is omitted from the H&E method.]; PA0 (i) Removing excess stain and water by a series of sequential washes in a dehydrating reagent; PA0 (j) Contacting the slide with a chemical-clearing agent (toluene, xylene, or t-butanol) to remove residual dehydrating reagent remaining from the washing step; PA0 (k) Applying a cover-slip mountant and a cover-slip after first removing the slide from the chemical-clearing agent. The clearing agent evaporates and the mountant hardens leaving a stained and mounted slide. PA0 1. In an H&E method for histology specimens, the hematoxylin in step (d) is usually more concentrated for tissue sections than for cytology specimens. Regressive hematoxylin stain usually employs a more concentrated hematoxylin. PA0 2. In the H&E method, steps (f) and (g) are omitted. PA0 3. The green component of the cytoplasmic stain in step (h) for cytology specimens is omitted when performing an H&E stain on histology specimens.
As seen from the above steps, the staining of cytology and histology specimens are very similar. However, for clarity's sake the following differences between the handling of histology specimens as compared to cytology specimens are noted:
There are several disadvantages to the prior art immersion staining techniques. The chemical-clearing agents typically employed in the chemical-clearing step include many toxic substances which can harm the environment and present a hazard to laboratory personnel. Furthermore, the immersion staining techniques usually take from fifteen to twenty minutes and include many washes, each performed several times. Still further, the open baths of volatile dehydrating reagents used lead to reagent loss due to vaporization of reagent. Dipping slides into a bath also lends itself to loss of reagents. Dipping in a bath also leads to contamination of the bath with the reagent of the previous step resulting in inefficient use of washing-dehydrating reagents. In addition, cells can come off the slide into the reagent bath and contaminate other specimens, i.e., cross-contamination of specimens.
In the past, the baths of washing-dehydrating reagent typically consisted of three increasingly concentrated ethanol solutions, typically 50%, 75%, and 95%. It was thought that initial exposure to a more dilute solution was needed to reduce shock to the specimen. Current practice is to start with primary, secondary, and tertiary baths of 95% ethanol so that the sequential baths becomes more and more diluted as the number of slides exposed to the bath increases. When the tertiary bath begins to show significant contamination with dye or otherwise becomes diluted it is used as the secondary bath, the secondary bath is then used as the primary bath, and a new, fresh, concentrated tertiary ethanol bath is employed. Current practice has a similar disadvantage to that of the prior practice in that it consists of exposing the slide to essentially a series of three reagent solutions, each increasingly more concentrated than its predecessor.
Improved staining methods for histology and cytology specimens, decreasing the number of steps and the number and quantity of reagents consumed has long been needed, but providing for same has remained a problem. Immersion staining has been automated. However, it has been done by merely adapting the hand staining procedure to be performed by machine without any change in the steps of the procedure. In the related field of staining bacterial specimens, the Gram stain method has been adapted to be performed on automated spray staining equipment, such as the Aerospray.RTM. stainer manufactured by Wescor, Inc. of Logan, Utah. However, although the automated spray method of performing the Gram stain solves the contamination problem, it does not teach or suggest how to decrease the number of steps and the number an quantity of reagents consumed in cytology and histology staining procedures.